Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

Abstract

ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D—and to a lesser extent also mutants S22D and S27D—are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-1M-I116) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (KD ∼10-fold higher than wild-type ACA8) and S99D (KD about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-1M-I116 peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed.

Quantitative phosphoproteomic analysis reveals common regulatory mechanisms between effector‐ and PAMP‐triggered immunity in plants

“…ACA8 forms a protein complex with FLS2 and is involved in flg22‐inducible Ca 2+ signaling (Frei dit Frey et al ., ). flg22 treatment/perception induces phosphorylation at ACA8 S22, and this N‐terminal phosphorylation is important for the interaction of ACA8 with CALMODULIN (CaM), leading to ACA8 activation (Giacometti et al ., ). We found that RPS2 activation similarly induced S22 phosphorylation (Tables , S4), suggesting CaM‐mediated activation of ACA8 during both PTI and ETI.…”

“…We found that RPS2 activation similarly induced S22 phosphorylation (Tables , S4), suggesting CaM‐mediated activation of ACA8 during both PTI and ETI. Interestingly, CPK16 can phosphorylate S22 of ACA8 in vitro (Giacometti et al ., ), suggesting a role of CPK16 in the PTI and ETI signaling pathways. CBL‐INTERACTING PROTEIN KINASE‐9 (CIPK9) in a complex with the plasma membrane Ca 2+ sensor CALCINEURIN B‐LIKE PROTEIN‐1 (CBL1) phosphorylates ACA8, thereby regulating its activity (Costa et al ., ).…”

“…In addition, we found commonly phosphorylated sites during PTI and ETI in PEN3 and ACA8. Importantly, previous mutational analyses have shown that these phosphorylation sites of PEN3 and ACA8 are required for their activation (Giacometti et al ., ; Underwood & Somerville, ).…”

Properties and functions of calcium‐dependent protein kinases and their relatives inArabidopsis thaliana

“…(), [10] Giacometti et al . (), [11] Curran et al . (), [12] Bohmer & Romeis (), [13] Boudsocq et al .…”

ACA12 is a deregulated isoform of plasma membrane Ca2+-ATPase of Arabidopsis thaliana

“…Since both BRI1 and CLV1 are members of the large family of receptor-like kinases (RLK) (Gish and Clark 2011), the two pumps might be substrate of their kinase activity. In the case of ACA8 it is known that this pump is phosphorylated in vivo at several Ser residues in the regulatory N-terminus and that phosphorylation can affect both auto-inhibition and CaM-binding (Giacometti et al 2012 and references therein). Although most of these Ser residues are not conserved in ACA12, we cannot exclude that phospho- dephosphorylation might also impact ACA12 activity, and that the yeast expressed enzyme might have properties different from that expressed in planta.…”